Yeast and filamentous fungi have both been successfully used for the production of recombinant proteins, both intracellular and secreted (Cereghino, J. L. and J. M. Cregg 2000 FEMS Microbiology Reviews 24(1): 45-66; Harkki, A., et al. 1989 Bio-Technology 7(6): 596; Berka, R. M., et al. 1992 Abstr. Papers Amer. Chem. Soc. 203: 121-BIOT; Svetina, M., et al. 2000 J. Biotechnol. 76(2-3): 245-251). Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha, have played particularly important roles as eukaryotic expression systems because they are able to grow to high cell densities and secrete large quantities of recombinant protein. Likewise, filamentous fungi, such as Aspergillus niger, Fusarium sp, Neurospora crassa and others, have been used to efficiently produce glycoproteins in industrial scale. However, glycoproteins expressed in any of these eukaryotic microorganisms differ substantially in N-glycan structure from those in animals. This has prevented the use of yeast or filamentous fungi as hosts for the production of glycosylated therapeutic proteins.
Currently, expression systems such as yeast, filamentous fungi, plants, algae and insect cell lines (lower eukaryotes) are being investigated for the production of therapeutic proteins, which are safer, faster and yield higher product titers than mammalian systems. These systems share a common secretory pathway in N-linked oligosaccharide synthesis. Recently, it was shown that the secretory pathway of P. pastoris can be genetically re-engineered to perform sequential glycosylation reactions that mimic early processing of N-glycans in humans and other higher mammals (Choi et al., Proc Natl Acad Sci USA. 2003 Apr. 29; 100(9):5022-7). In addition, production of human glycoproteins with complex N-glycans lacking galactose through re-engineering the secretory pathway in yeast P. pastoris has been shown (Hamilton et al., Science. 2003 Aug. 29; 301(5637):1244-6). In mammalian cells, further maturation involves galactose transfer. Consequently, the maturation of complex glycosylation pathways from yeast and lower eukaryotes requires the functional expression of β1,4-galactosyltransferase.
Recombinant expression of UDP-Gal: βGlcNAc β1,4-galactosyltransferase (β1,4GalT) has been demonstrated in mammalian cells, insect cells (e.g., Sf-9) and yeast cells. A cDNA encoding a soluble form of the human β1,4-galactosyltransferase I (EC 2.4.1.22) (lacking the endogenous Type II membrane domain) has also been expressed in the methylotrophic yeast P. pastoris. Malissard et al. Biochem Biophys Res Commun. 2000 Jan. 7; 267(1):169-73. Additionally, gene fusions encoding ScMnt1p fused to the catalytic domain of a human β1,4-galactosyltransferase (Gal-Tf) have been expressed showing some activity of the enzyme in the yeast Golgi albeit at very low conversion efficiency. Schwientek et al., J Biol. Chem. 1996 Feb. 16; 271(7):3398-405. Thus, targeting a β1,4-galactosyltranferase (β1,4GalT) to the secretory pathway of a host that produces glycans containing terminal GlcNAc is expected to result in some galactose transfer. However the formation of complex glycans in higher eukaryotes involves the action of mannosidase II which in mammalian cells has been found to act in competition with GalTI (Fukuta et al., Arch Biochem Biophys. 2001 Aug. 1; 392(1):79-86). The premature action of GalT is thus expected to prevent the formation of complex galactosylated glycoproteins in the secretory pathway and yield mostly hybrid glycans.
The N-glycans of mammalian glycoproteins typically include galactose, fucose, and terminal sialic acid. These sugars are not usually found on glycoproteins produced in yeast and filamentous fungi. In humans, nucleotide sugar precursors (e.g. UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose, etc.) are synthesized in the cytosol and transported into the Golgi, where they are incorporated into N-glycans by glycosyltransferases (Sommers and Hirschberg, 1981 J. Cell Biol. 91(2): A406-A406; Sommers and Hirschberg 1982 J. Biol. Chem. 257(18): 811-817; Perez and Hirschberg 1987 Methods in Enzymology 138: 709-715).
Glycosylation engineering in heterologous protein expression systems may involve expression of various enzymes that are involved in the synthesis of nucleotide sugar precursors. The enzyme UDP-galactose 4-epimerase converts the sugar nucleotide UDP-glucose to UDP-galactose via an epimerization of C4. The enzyme has been found in organisms that are able to use galactose as its sole carbon source. Recently, the bifunctional enzyme, Gal10p, has been purified in Saccharomyces cerevisiae having both a UDP-glucose 4-epimerase and aldose 1-epimerase activity. Majumdar et al., Eur J. Biochem. 2004 February; 271(4):753-759.
The UDP-galactose transporters (UGT) transport UDP-galactose from the cytosol to the lumen of the Golgi. Two heterologous genes, gmal2(+) encoding alpha 1,2-galactosyltransferase (alpha 1,2 GalT) from Schizosaccharomyces pombe and (hUGT2) encoding human UDP-galactose transporter, have been functionally expressed in S. cerevisiae to examine the intracellular conditions required for galactosylation. Correlation between protein galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter, played a key role for efficient galactosylation in S. cerevisiae (Kainuma, 1999 Glycobiology 9(2): 133-141). Likewise, a UDP-galactose transporter from S. pombe was cloned (Aoki, 1999 J. Biochem. 126(5): 940-950; Segawa, 1999 Febs Letters 451(3): 295-298).
Glycosyltransfer reactions typically yield a side product which is a nucleoside diphosphate or monophosphate. While monophosphates can be directly exported in exchange for nucleoside diphosphate sugars by an antiport mechanism, diphosphonucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphates and inorganic phosphate prior to being exported. This reaction is important for efficient glycosylation; for example, GDPase from S. cerevisiae has been found to be necessary for mannosylation. However that GDPase has 90% reduced activity toward UDP (Berninsone et al., 1994 J. Biol. Chem. 269(1):207-211). Lower eukaryotes typically lack UDP-specific diphosphatase activity in the Golgi since they do not utilize UDP-sugar precursors for Golgi-based glycoprotein synthesis. S. pombe, a yeast found to add galactose residues to cell wall polysaccharides (from UDP-galactose) has been found to have specific UDPase activity, indicating the potential requirement for such an enzyme (Berninsone et al., 1994).
UDP is known to be a potent inhibitor of glycosyltransferases and the removal of this glycosylation side product may be important to prevent glycosyltransferase inhibition in the lumen of the Golgi (Khatara et al., 1974). See Berninsone, P., et al. 1995. J. Biol. Chem. 270(24): 14564-14567; Beaudet, L., et al. 1998 Abc Transporters: Biochemical, Cellular, and Molecular Aspects. 292: 397-413.
What is needed, therefore, is a method to catalyze the transfer of galactose residues from a sufficient pool of UDP-galactose onto preferred acceptor substrates for use as therapeutic glycoproteins.